Work in the present grant period developed a new hydrogen exchange method that appears to define three intermediates in the cytochrome c folding pathway. Also kinetic experiments were done to define the barriers that cytochrome c must overcome in their folding trajectory. The information obtained points to a coherent set of steps that may describe the major pathway in cytochrome c folding. The proposed work is designed to test and build on these hypotheses, which can be listed as follows. 1) The unfolded state: Under normal solution conditions, unfolded polypeptides exist as non-specifically contracted chains. In stopped-flow folding experiments that start by diluting unfolded proteins from high denaturant, this biased U condition is reached very rapidly (less than 1 msec), and does not represent a productive folding intermediate. 2) Nucleation: Folding begins with and initial energetically uphill large scale conformational search to reach a transition state nucleus that can support forward folding steps in a downhill manner, and therefore must be native-like in some topological sense. This diffusional search-dependent step typically requires 1 to 10 msec. 3) Folding intermediates: From the folding nucleus, the cyt c chain moves energetically downhill through a sequence of three increasingly native-like, cooperatively structured intermediates to the native state. The step from one intermediate to the next requires only a fast, small scale conformational search. 4) Error barriers: In this downhill sequence the refolding protein may, often with high probability, encounter one or more optional, error-dependent misfold- reorganization barriers that can slow native state acquisition, typically (for small proteins) to the approximate 1 second time scale. These hypotheses will be tested by applying our earlier hydrogen exchange pulse labeling method, our newer native state hydrogen exchange method. 2D NMR, other spectroscopies (CD, fluorescence, absorbance), and rapid reaction methods (stopped-flow). The experiments will use mammalian, bacterial, and genetically engineered versions of cytochrome c as model proteins, and other useful model proteins.